UV protective skin treatment compositions and screening methods

ABSTRACT

Provided are methods of screening antioxidants for skin protective capacity and antioxidant formulations which protect the skin from UV radiation. The method of screening antioxidants for protecting the skin from UV radiation includes using the Briggs-Rauscher Oxidant Method (BROM) method to one or more antioxidants for predicting protection to the epidermal layer. One advantageous antioxidant formulation for use in sun screens, lip balms and other types of topically applied skin care products for protecting the skin from UV radiation includes a mixture of antioxidants in the skin care product including an effective amount of arbutin in combination with BHT, wherein the ratio of arbutin to BHT in the skin care product ranges from 1:4 to 4:1, and wherein the measured BROM value of the antioxidant mixture is greater than the sum of the BROM values of the individual antioxidants in the mixture at the same concentration. The measured BROM value of the resultant product is greater than or equal to 0.2 sec/μg. The antioxidant formulation may also optionally include hydroquinone, resveratrol, uric acid and/or atorvastatin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application U.S. patent applicationSer. No. 13/544,206, filed on Jul. 9, 2012, and claims the benefit ofU.S. Provisional Patent Application No. 61/506,394 filed on Jul. 11,2011. The disclosures of which are incorporated herein by reference intheir entireties.

FIELD

The present disclosure relates to the field of antioxidant compositionsfor protection against cellular damage to the layers of the skin fromfree radicals generated from sunlight exposure or during metabolicprocesses in the human body. It also relates to novel uses of screeningmethods for evaluating antioxidants for their protective capacity to thedifferent layers of the skin. More particularly, the present disclosurerelates to novel combinations of antioxidants that have enhancedantioxidant capacity as measured by the Briggs-Rausher OscillationReaction Method (BROM) and the use of such antioxidants inpharmaceutical or dermatological carrier compositions for enhancedantioxidant capacity.

BACKGROUND

The skin of the human body is an organ that acts as a protectiveinterface with the ambient environment. The epidermis is the outermostlayer of the skin. It forms a waterproof, protective wrap over thebody's and lips' surfaces and is made up of stratified squamousepithelium which is an underlying basal lamina. The epidermis containsno blood vessels, and cells in the deepest layer obtain nutrients bydiffusion from the blood capillaries extending to the upper layers ofthe dermis. The main type of cells which comprise the epidermis areMerkel cells, keratinocytes with melanocytes and Langerhans cells. Theepidermis can be further subdivided into the following strata (beginningwith the outermost layer):corneum, lucidum (only in palms of hands andbottoms of feet), granulosum, spinosum, and basale. Cells are formedthrough mitosis at the basal layer. The daughter cells move up thestrata by changing shape and composition as they die due to isolationfrom their blood source. The cytoplasm is released and protein keratinis inserted. The cells move up to the corneum and slough off throughdesquamation, which is a process called keratinization taking place overa 27-day period. This keratinized layer of skin and lips is responsiblefor keeping water in the body and keeping other harmful chemicals andpathogens out while providing a natural barrier to infection. Theexternal layers of the skin change in the number of cellular layers, andwhile, for example, the facial skin has sixteen cellular layers, thespecialized keratinized outer mucosa of the lips only has three to fivecellular layers. Always the common factor is that the outer cellularlayers are very low in water (circa 20%), while the deeper layers have amuch higher water content (circa 80%).

The skin is exposed to several forms of stress including ozone,ultraviolet (UV) radiation, air pollution, pathologic microorganisms,chemical oxidants and topically applied substances. Oxidative stressoccurs when some molecules (oxidizing agents) take electrons from theother molecules or atoms. The substances that can exist with missingelectrons are called free radicals. Most of these free radicals areoxygen molecules or atoms. Free radicals are highly reactive in thatthey are ready to give away an electron or to accept one. After thelonesome electron pairs, the atom loses its radical activity, but theatom that has just lost an electron becomes a free radical in turn.These free radical reactions are a necessary part of metabolicprocesses, however, too many free radicals cause dangerous chainreactions that may destroy cellular composition and may cause damage toDNA, skin proteins and lipids (fats) through these oxidative stresses.The skin of the human body is exposed to oxidative stressors through theenvironment, bi-products of metabolism, and lifestyle factors such assmoking, alcohol, and UV radiation exposure. These oxidative stressesstimulate the production of unstable molecules otherwise known asreactive oxygen species (ROS) or free radicals. Free radicals are highlyreactive molecules that are created as bi-products of normal metabolism(intrinsic) and environmental stressors (extrinsic), and are responsiblefor cellular damage to the skin. Cellular damage to the layers of theskin can be from free radicals generated from sun light exposure orduring metabolic processes in the human body.

Sunlight in a broad sense refers to the total frequency spectrum ofelectromagnetic radiation given off by the sun. The solar spectrumconsists of electromagnetic radiation with a wavelength ranging from 200to 2500 nm, which includes UV, visible and infrared radiation. The UVportion of the spectrum (200 to 400 nm) is responsible for the mostdamage to human skin. The UV spectrum may be further broken down by thelong wave portion (UVA), and a medium wave portion (UVB). Table 1 belowprovides a breakdown of the wavelength range and energy per photon forthe UVA and UVB portions of the ultraviolet spectrum.

TABLE 1 Wavelength range Energy Name Abbreviation in nanometers perphoton* Ultraviolet A, long wave, UVA 400 nm-315 nm 3.10-3.94 eV orblack light Ultraviolet B or medium UVB 315 nm-280 nm 3.94-4.43 eV wave*1 eV = 1.6 × 10−19 joule

The UV flux at the Earth's surface has been determined and the erythemaldose to human skin at local noon has been calculated for all latitudesand it varies from 0.0 to 0.4 W/m². Because of variations in theintensity of UV radiation passing through the atmosphere, the risk ofsunburn increases with proximity to the tropic latitudes, locatedbetween 23.5° north and south latitude. The noon erythemal dose to humanskin at 23.5° latitude is 0.25 W/m². FIG. 1 shows the depth ofpenetration for UVA and UVB radiation through the skin. From FIG. 1, itcan be seen that the sun burning radiation (wavelengths shorter than 315nanometers) is nearly all absorbed in the epidermis, which is where UVBradiation has it most harmful effects. Wavelengths longer than 315nanometers (UVA) is nearly all absorbed in the epidermis and dermis,which is where UVA radiation has it most harmful effects.

Upon absorption of UV radiation energy by photo reactive molecules(chromophore), a photo chemical reaction is induced. The absorption ofthe radiation energy by the chromophores (P) in its ground state willoccur. The formation of the excited (usually triplet) state (³P)molecule occurs. The excited state molecules then participate in oxygendependent processes (i.e., photodynamic processes) in two majorpathways: Type I or Type II reactions, both of which result in cytotoxicinjury to the skin. The cytotoxic injury by the UV radiation causesmutations and death in cells of the skin. The Type I reaction involvestransfer of an electron or a hydrogen atom to the excited statephotosensitizer (³P) resulting in the formation of free radicals(Equation 1 below), which leads to oxidative reduction reactions thatresults in hydrogen peroxide formation and subsequent cell damage(Equations 2 and 3 below).³P+RH→PH.+R.  (1)PH.+PH.→P+PH₂  (2)PH₂+O₂→P+H₂O₂  (3)³P+O₂→P+¹O₂  (4)

The interaction of ³P with ground state oxygen could result in theformation of superoxide anions (O₂.)—, which in turn, can be convertedinto highly reactive and cytoxic hydroxyl radicals (OH.). The Type IIreaction depicted above is also known as an energy transfer process.Transfer of energy to ground state oxygen results in the formation ofsinglet oxygen (¹O₂), which is highly reactive and has a lifetime of 50nanoseconds (See equation 4 above). Cytotoxic injury occurs upon singletoxygen-induced oxidation of amino acids and unsaturated fatty acids withinteraction of the latter resulting in the formation of hydroperoxides,which initiate lipid and protein oxidation.

Exposure to the UV spectrum of sunlight through the mechanisms describedabove has been associated with skin cancer. Excessive UV radiation fromsun exposure causes DNA damage, inflammation, erythema, sunburn,immunosuppression, photoaging, gene mutations, and skin cancer. Sunburncan also be caused by pharmaceutical products that sensitize some usersto UV radiation. Certain antibiotics, oral contraceptives, andtranquilizers have this effect. In general, people with fair hair and/oralso freckles have a greater risk of sunburn than others because oftheir lighter skin tone and low melanin in the skin. Upon DNA damage,tumor suppressor protein undergoes phosphorylation and translocation tothe nucleus and aids in DNA repair or causes apoptosis. Excessive UVexposure overwhelms DNA repair mechanisms. UV radiation is a commoncause of melanoma and sunburn. Sunburn is caused by direct DNA-damage,whereas melanoma is caused by indirect DNA-damage. Protecting againstsunburn does not necessarily protect against melanoma, howeverprotection of the skin from sunlight radiation is highly desirable. Thepreferred skin protection method against UV radiation damage isclothing, including hats. Moderate sun tanning without burning can alsoprevent subsequent sunburn, as it increases the amount of melanin, askin photoprotectant pigment that is the skin's natural defense againstoverexposure. The erythemal diurnal dose rate for sun tanning isdependent on latitude and time of the solar day.

Sunscreens can help prevent sunburn, although they may not effectivelyprotect against malignant melanoma, which either is caused by adifferent part of the UV spectrum or is not caused by sun exposure atall. Sunscreens utilize a combination of antioxidants, which aregenerally selected haphazardly without taking into consideration thephysicochemical factors which will help prevent cytotoxic damage. Theuse of sunscreen is known to prevent the direct DNA damage that causessunburn and the two most common forms of skin cancer, basal-cellcarcinoma and squamous cell carcinoma. However, if sunscreen penetratesinto the skin, it promotes indirect DNA damage, which causes the mostlethal form of skin cancer, malignant melanoma. The existing SPF labelvalue for sunscreen is based according to the formula SPF/MinimalErythemal Dose ⅔ where the effective erythemal dose is 200 W/m². At adose rate 0.250 W/m² it takes 800 seconds of local noon exposure at alatitude of 23° N (Key West, Fla.) to be exposed to 1 MED of UVradiation. In addition to UV mediated photosensitization, the sunscreenfilter is absorbed into the skin, and prevents sunburn, but alsoincreases the amount of free radicals, which in turn increases the riskfor malignant melanoma. The harmful effect of photo-excited sunscreenfilters on living tissue has been shown in many photo-biologicalstudies. Whether sunscreen prevents or promotes the development ofmelanoma depends on the relative importance of the protective effectfrom the topical sunscreen versus the harmful effects of the absorbedsunscreen. Therefore, it is highly desirable to develop compositions ofantioxidants that will prevent UV induced oxidative damage to the outerlayers of the skin.

In order to determine the various physiological effects of UV radiationon human skin, it is necessary to develop an understanding of the depthof penetration of the different UV wavelengths into the skin. But thisis difficult because the skin is made up of optically inhomogeneouslayers having different properties and varying in thickness andstructure from one part of the body to another. An understanding of thedifferent reaction mechanisms taking place with the skin from UVradiation and how the structure and chemical composition of skin variesfrom these UV induced oxidative stresses is important to developing skintreatment compositions that protect the skin from harmful UV radiation.Therefore, there is also a need to develop methods that aid in thescreening and selection of antioxidant combinations that may be used toprotect the skin from the harmful effects of UV radiation and otherenvironmental and metabolic processes.

SUMMARY

According to the present disclosure, an advantageous method of screeningantioxidants for protecting the outer layers of the skin from UVradiation includes the use of the Briggs-Rauscher Oxidant Method(hereinafter BROM) for predicting antioxidants for protection to theepidermal layers and to help minimize the damage caused byphotodecomposition of exogenous materials that penetrate the surface ofthe skin while being applied for cosmetic or pharmaceutical purposes.

A further aspect of the present disclosure relates to advantageousantioxidant formulations for topically applied skin care products forprotecting the skin from direct and indirect effects of UV radiation.The direct effects include the photodynamic processes previouslydescribed and the indirect effects include the phototoxicity generatedby the photodecomposition of exogenous products present on the skin,e.g., treatment products, sunscreens and/or medicaments. A list ofpreferred antioxidants for this purpose are those that individually orin combinations, have sufficiently large BROM values to overcome freeradical cellular photo damage induced by the radiative photon flux.

In one form of the present disclosure, an antioxidant formulation foruse in sun screens, lip balms and other topically applied skin careproducts for protecting the skin from UV radiation includes: a mixtureof antioxidants in the skin care product including an effective amountof arbutin and BHT, wherein the ratio of arbutin to BHT in the skin careproduct ranges from 1:4 to 4:1, and wherein the measured BROM value ofthe antioxidant mixture is greater than the sum of the BROM values ofthe individual antioxidants in the mixture at the same concentration.

In another form of the present disclosure, antioxidant formulation foruse in sun screens, lip balms and other topically applied skin careproducts for protecting the skin from UV radiation includes: a mixtureof antioxidants in the skin care product including an effective amountof atorvastatin and BHT, wherein the ratio of atorvastatin to BHT in theskin care product ranges from 1:2 to 1:1, and wherein the measured BROMvalue of the antioxidant mixture is greater than the sum of the BROMvalues of the individual antioxidants in the mixture at the sameconcentration.

In another form of the present disclosure, an antioxidant formulationfor use in sun screens, lip balms and other topically applied skin careproducts for protecting the skin from UV radiation includes: a mixtureof antioxidants in the skin care product including an effective amountof arbutin and hydroquinone, wherein the ratio of arbutin tohydroquinone in the skin care product ranges from 1:4 to 4:1, andwherein the measured BROM value of the antioxidant mixture is greaterthan the sum of the BROM values of the individual antioxidants in themixture at the same concentration.

In another form of the present disclosure, an antioxidant formulationfor use in sun screens, lip balms and other topically applied skin careproducts for protecting the skin from UV radiation includes: a mixtureof antioxidants in the skin care product including an effective amountof hydroquinone and BHT, wherein the ratio of hydroquinone to BHT in theskin care product ranges from 1:4 to 4:1, and wherein the measured BROMvalue of the antioxidant mixture is greater than the sum of the BROMvalues of the individual antioxidants in the mixture at the sameconcentration.

In yet another form of the present disclosure, an antioxidantformulation for use in sun screens, lip balms and other topicallyapplied skin care products for protecting the skin from UV radiationincludes: a mixture of antioxidants in the skin care product includingan effective amount of atorvastatin and uric acid, wherein the ratio ofatorvastatin to uric acid in the skin care product ranges from 0.5:1 to2:1, and wherein the measured BROM value of the antioxidant mixture isgreater than the sum of the BROM values of the individual antioxidantsin the mixture at the same concentration.

In yet another form of the present disclosure, an antioxidantformulation for use in sun screens, lip balms and other topicallyapplied skin care products for protecting the skin from UV radiationincludes: a mixture of antioxidants in the skin care product includingan effective amount of uric acid, atorvastatin and BHT, wherein theratio of uric acid, atorvastatin and BHT in the skin care product rangesfrom 1:1:4 to 4:1:1, and wherein the measured BROM value of theantioxidant mixture is greater than the sum of the BROM values of theindividual antioxidants in the mixture at the same concentration.

In yet another form of the present disclosure, an antioxidantformulation for use in sun screens, lip balms and other topicallyapplied skin care products for protecting the skin from UV radiationincludes: a mixture of antioxidants in the skin care product includingan effective amount of arbutin and resveratrol, wherein the ratio ofarbutin to resveratrol in the skin care product ranges from 1:4 to 4:1,and wherein the measured BROM value of the antioxidant mixture isgreater than the sum of the BROM values of the individual antioxidantsin the mixture at the same concentration.

In still yet another form of the present disclosure, an antioxidantformulation for use in sun screens, lip balms and other topicallyapplied skin care products for protecting the skin from UV radiationincludes: a mixture of antioxidants in the skin care product includingan effective amount of BHT and resveratrol, wherein the ratio of BHT toresveratrol in the skin care product ranges from 1:4 to 4:1, and whereinthe measured BROM value of the antioxidant mixture is greater than thesum of the BROM values of the individual antioxidants in the mixture atthe same concentration.

In still yet another form of the present disclosure, an antioxidantformulation for use in sun screens, lip balms and other topicallyapplied skin care products for protecting the skin from UV radiationincludes: a mixture of antioxidants in the skin care product includingan effective amount of atorvastatin and hydroquinone, wherein the ratioof atorvastatin to hydroquinone in the skin care product ranges from 1:4to 4:1, and wherein the measured BROM value of the antioxidant mixtureis greater than the sum of the BROM values of the individualantioxidants in the mixture at the same concentration.

In still yet another form of the present disclosure, an antioxidantformulation for use in sun screens, lip balms and other topicallyapplied skin care products for protecting the skin from UV radiationincludes: a mixture of antioxidants in the skin care product includingan effective amount of arbutin, hydroquinone and BHT, wherein the ratioof arbutin, hydroquinone and BHT in the skin care product ranges from2:1:1 to 2:1:4, and wherein the measured BROM value of the antioxidantmixture is greater than the sum of the BROM values of the individualantioxidants in the mixture at the same concentration.

In still yet another form of the present disclosure, an antioxidantformulation for use in sun screens, lip balms and other topicallyapplied skin care products for protecting the skin from UV radiationincludes: a mixture of antioxidants in the skin care product includingan effective amount of arbutin, BHT and resveratrol, wherein the ratioof arbutin, BHT and resveratrol in the skin care product ranges from1:1:1 to 2:1:2, and wherein the measured BROM value of the antioxidantmixture is greater than the sum of the BROM values of the individualantioxidants in the mixture at the same concentration.

These and other features and attributes of the disclosed methods forscreening skin protective antioxidants from UV radiation, and theantioxidant formulations for protecting the skin from UV radiation ofthe present disclosure will be apparent from the detailed descriptionwhich follows, particularly when read in conjunction with the figuresappended hereto.

BRIEF DESCRIPTION OF DRAWINGS

To assist those of ordinary skill in the relevant art in making andusing the subject matter hereof, reference is made to the appendeddrawings, wherein:

FIG. 1 depicts a schematic of the depth of penetration for UVA and UVBradiation through the epidermal and dermal layers of the skin.

FIG. 2 is a bar graph of % cytotoxicity relative to a control of Sample1 (JR-42-24C) as a function of concentration of JR-42-24C in thesolution.

FIG. 3 is a bar graph of % photocytotoxicity relative to a control ofSample 1 (JR-42-24C) as a function of concentration of JR-42-24C in thesolution.

FIG. 4 is a bar graph of % cytotoxicity relative to a control of Sample2 (JR-42-26A) as a function of concentration of JR-42-26A in thesolution.

FIG. 5 is a bar graph of % photocytotoxicity relative to a control ofSample 2 (JR-42-26A) as a function of concentration of JR-42-26A in thesolution.

FIG. 6 is a bar graph of % cytotoxicity relative to a control of Sample3 (JR-42-26C) as a function of concentration of JR-42-26C in thesolution.

FIG. 7 is a bar graph of % photocytotoxicity relative to a control ofSample 3 (JR-42-26C) as a function of concentration of JR-42-26C in thesolution.

DETAILED DESCRIPTION

The present disclosure provides novel methods for predicting theperformance of antioxidants in sunscreens and other topically appliedskin products and novel antioxidant combinations for use in suchsunscreens and other topically applied skin products. All numericalvalues within the detailed description and the claims herein aremodified by “about” or “approximately” the indicated value, and takeinto account experimental error and variations that would be expected bya person having ordinary skill in the art.

UVA-irradiation was found to generate cyclobutane dimers at TT and to alower extent at TC sites as a likely result of energy transfer mechanisminvolving still unknown photoexcited chromophore(s). Oxidative damage toDNA is also induced although less efficiently by UVA-mediatedphotosensitization processes than UVB. UVA mostly involves singletoxygen species together with a smaller contribution of hydroxylradical-mediated reactions through initially generated superoxideradicals.

A radical chain reaction inhibitor is commonly regarded as anantioxidant. The human skin deploys a rich complement of antioxidantmechanisms against various forms of oxidative stress. These biologicalantioxidant mechanisms can be broken down by enzymatic andnon-enzymatic. Enzymatic mechanisms include, but are not limited to,superoxide dismutase, catalase and gluthione peroxidase. Non-enzymaticmechanisms include, but are not limited to, antioxidant enzyme cofactors(Se, coenzyme Q10), oxidative enzyme inhibitor (aspirin ibuprofen),transmission metal chelators (EDTA) and radical scavengers (such asVitamin C and E).

Reactive oxygen species (ROS) are responsible for oxidative stress. Ithas been discovered that there are six major reactive oxygen speciescausing oxidative damage in the human body. These species are superoxideanion (O₂.), hydrogen peroxide (H₂O₂.), peroxyl radicals (ROO.),hydroxyl radical (HO.), singlet oxygen (¹O₂.), and peroxynitrate(ONOO.). To counteract the assault of these ROS, living cells have abiological defense system composed of enzymatic antioxidants thatconvert ROS/RNS to harmless species. For example super oxide ion (O₂.)⁻is converted to oxygen by catalase. In contrast, no enzymatic action isknown to scavenge ROO., HO., ¹O₂., and ONOO.. Therefore, the burden ofdefense relies on a variety of nonenzymatic antioxidants, such asvitamins C and E and many chemicals, which have the property ofscavenging oxidants and free radicals. To comprehensively evaluate theoxidant-scavenging capacity of a sample, assays have to be designed toinclude these ROS. However, the majority of assays are designed tomeasure a sample's capacity to react with one oxidant (either organicradical or redox active compounds). The peroxyl radical has been themost frequently used ROS in the assays because it is a key radical inauto-oxidation and it can be easily generated.

A multitude of in vitro antioxidant capacity assays have been developed.The application of these in vitro assays in clinical research arelimited due to the following: from being based on chemical reactions invitro, though similarly to biological systems that can only be impliedby the reaction mechanism involved, through the pH of the media, andthrough the standard redox potentials that are similar to those involvedin cellular reactions. Non-limiting exemplary in vitro antioxidantcapacity assays include the ORAC (oxygen radical absorbance capacity),the TRAP (total radical trapping antioxidant parameter), the Crocinbleaching assay, the IOU (inhibited oxygen uptake), the inhibition oflinoleic acid oxidation, the inhibition of LDL oxidation, the TEAC(Trolox equivalent antioxidant capacity), the FRAP (ferric ion reducingantioxidant capacity), the DPPH (diphenyl-1-picrylhydrazyl), the copper(II) reduction capacity, the total phenols assay by Folin-Ciocalteureagent, the TOSC (total oxidant scavenging capacity),chemiluminescence, electochemiluminescence, and the inhibition ofBriggs-Rauscher oscillation method (BROM).

It is problematic to apply antioxidant capacity assays to aninhomogeneous biological substrate like the human skin. The epidermiscontains no blood cells and only the cells in the deeper layer receiveblood by diffusion from blood capillaries that extend to the upperlayers of the dermis. Also the concentration gradient of water contentvaries from very low water content in the stratum corneum to higherwater content in the basal layer of the epidermis. The oversimplifieddivision of antioxidants in water soluble or lipid soluble is not reallyuseful of their specific roles in the chemical kinetics of skin cellulardamage. The basic kinetic models of inhibited auto-oxidation depend onthe chemical reactions involved. Some compounds contribute to theantioxidant defense by scavenging the free radicals while others bytheir ability to chemically reduce the oxidants present.

The mechanism of reactions for antioxidant capacity are divided in twotypes of kinetics, hydrogen atom transfer (HAT) and electron transfer(ET). The majority of HAT-based assays apply a competitive reactionscheme, in which antioxidant and substrate compete for thermallygenerated peroxyl radicals. In contrast, ET-based assays measure thecapacity of an antioxidant in the reduction of an oxidant, when reduced.The degree of antioxidant capacity is correlated with the sample'santioxidant concentrations. ET-based assays include, but are not limitedto, the Trolox equivalence antioxidant capacity, the ferric ion reducingantioxidant power (FRAP), and the Briggs-Rausher Oscillation ReactionMethod (BROM).

The Applicant has discovered the unexpected benefits of using anET-based in-vitro antioxidant assay in predicting the performance ofantioxidants in protecting the outer layers of human skin from theharmful effects of UV radiation. The BROM (Briggs-Rauscher) oscillationsare potentiometrically followed by using a bright platinum electrodecoupled with a suitable reference electrode. The main intermediates forwhich concentrations oscillate in the BR reaction are iodine; iodineion; oxyiodine species HOI, HOIO, and IO₂; and the hydroperoxyl radicalHOO.. In the onset of oscillations have been previously recognized inthe chemical literature. The assay method based on the inhibitoryeffects of antioxidants on the oscillations of hydrogen peroxide inducedfree radicals has been discovered by the Applicant to be predictive ofwhich antioxidants may be more effective protecting the shallow layersof the skin (epidermal layer) with low water content against the harmfuleffects of UV radiation. The BROM has been discovered by the Applicantto provide closer kinetic requirements to those present in differentlayers of the skin. The BROM method is relatively new in thedetermination of antioxidant capacity of many chemical compounds. Thesomewhat apparently low values for “well known” antioxidants likeVitamin E (0 oscillations sec/ug) and vitamin C (0.00105 sec/ug) can beeasily explained by the reaction conditions, and how the kinetictransfer associated with the method works.

The BROM method is used for the selection of preferred antioxidants tobe used in the upper layers of the skin with low water content. The BROMelectron transfer method takes place at a redox of 500 mV in acidconditions and antioxidant capacity can be determined in solvents withlittle water content. The BROM method detects the electron transfercurrent present where antioxidants and hydrogen peroxide are allowed toreact and has been discovered to be predictive of the electrophilicnature of antioxidants that act as electron or photon traps in the upperlayers of the skin.

Table 2 below gives the BROM values for common antioxidants present inbody fluids and tissues.

TABLE 2 Concen- tration Concentration in in liver Antioxidant humanserum tissue BROM metabolite Solubility (μM) (μmol/kg) [s/μg] AscorbicWater 50-60 260 (human) 0.00105 acid (vitamin C) Glutathione Water 46,400 0.000991 (human)¹ Uric acid Water 200-400 1,600 9.14 (human)Carotenes Lipid (β-carotene: 0.5-1 5 (human, 0 retinol (vitamin total(Tretinoin) A): 1-3 carotenoids) α-Tocopherol Lipid 10-40 50 (human) 0(vitamin E) Ubiquinol Lipid 5 200 (human) 0 (coenzyme Q10)

In Table 3 below, the BROM antioxidant capacity for some of theantioxidant metabolites listed in Table 2 and some other additionalantioxidant compounds are listed from lowest to highest values.

TABLE 3 Antioxidants by BROM strength BROM by Strength Sample ResultSize [μg] Solvent [s/μg] Allantoin 7700 Water 0 D-Glucose 700000 Water 0Oxybenzone 7500 Ethanol 0 Solastay S1 1600 Ethanol 0 Tretinoin 1700Ethanol 0 Vitamin E Acetate 33000 Ethanol 0 Ascorbyl ≈1300 Water Noclear Glucoside result Triethanolamine 2500 Water No clear Salicylateresult Glutathione 12000-15000 Water 0.000991 Ascorbic Acid 10000-100000 Water 0.00105 Trolox 1000-6000 Ethanol 0.021 Tert-1200-1900 Ethanol 0.13 Butylhydroquinone Grapefruit Peel  300-1300 50%0.38 Ethanol Hydroquinone 40582 50% 1.1 Ethanol Hydroquinone 40582 Water1.31 Hesperidin 40735 Water 5.38 Polyphenols 40672 Water 6.52Atorvastatin 22-32 Ethanol 8.41 (Lipitor) Uric Acid 130-180 Water 9.14Resveratrol 40609 Ethanol 18.34 BHT 40639 Ethanol 29.4 Resorcinol 40742Water 30.39 BHT 40645 50% 34.53 Ethanol β Arbutin 40739 Water 40.53Arbutin 40711 50% 47 Racemic mixture Ethanol Arbutin 40678 Water 49.4Racemic mixture

The results indicate that arbutin, butylatedhydroxytoluene (BHT),resorcinol, resveratrol, atorvastatin, uric acid, polyphenols, andhesperidin all have BROM values greater than 5 s/μg. The results ofTable 3 are unexpected in that well known antioxidants like vitamin C(ascorbic acid), Trolox (vitamin E derivative) and vitamin E acetate donot show measurable electron scavenging capacity. The results alsoindicate that only those antioxidants with electrophilic groups will beeffective in the upper layers of the skin where the photon induceddamage occurs in low water content media and no reducing conductivityoccurs. Therefore, it has been discovered that preferred antioxidants toact as free radical scavengers are molecules with highly electrophilicfunctional groups. The highest BROM values were obtained for certainsubstituted phenolic or polyphenolic compounds with aromatic rings thatcould easily react with unpaired electrons. Based on BROM values listedabove, it is predicted that the selected agents for better protectionagainst phototoxic free radical molecules should be the substitutedphenolics, such as arbutin and butylatedhydroxytoluene (BHT). However,antioxidants with BROM capacities greater than 5 sec/ug will havesimilar effects when added in sufficient concentrations to provide theconcentration threshold of antioxidants needed.

The Applicant has also discovered that various combinations ofantioxidants are effective for use in sun screens, lip balms and othertopically applied skin care products for protecting the skin from UVradiation. These antioxidant combinations include an effective amount oftwo, or three, or four or more individual antioxidants, wherein aneffective amount is defined as a concentration of antioxidants in theskin care product that results in a measured BROM value of theantioxidant mixture that is greater than the sum of the BROM values ofthe individual antioxidants in the mixture at the same concentration.When the antioxidant mixture in the product includes two antioxidants,the relative ratio of the two may be from 1:4 to 4:1, or 1:3 to 3:1, or1:2 to 2:1, or 1:2 to 1:1, or 1:1. When the antioxidant mixture in theproduct includes three antioxidants, the relative ratio of the three maybe from 1:1:4 to 4:1:1, or 1:1:3 to 3:1:1, or 1:1:2, to 2:1:1, or 2:1:1to 2:1:4, or 1:1:1 to 2:1:2. The following antioxidant mixturesincluding two antioxidants have been found to yield a synergistic impacton the measured BROM values, that is, the measured BROM value of theantioxidant mixture in the product is greater than the sum of the BROMvalues of the individual antioxidants in the mixture at the sameconcentration:arbutin/BHT, atorvastatin/BHT, arbutin/hydroquinone,hydroquinone/BHT, atorvastatin/uric acid, arbutin/resveratrol,BHT/resveratrol, and atorvastastin/hydroquinone. These antioxidantmixtures, which include two antioxidants, may also optionally include athird antioxidant (which is different from the first two) at aneffective amount to further reduce the measured BROM value of themixture relative to the sum of the BROM values of the individualantioxidants in the mixture at the same concentration, wherein the thirdantioxidant is chosen from atorvastatin, BHT, hydroquinone, resveratrol,arbutin, uric acid and combinations thereof.

The following antioxidant mixtures including three antioxidants havebeen found to yield a synergistic impact on the measured BROM values,that is, the measured BROM value of the antioxidant mixture in theproduct is greater than the sum of the BROM values of the individualantioxidants in the mixture at the same concentration:uricacid/atorvastatin/BHT, arbutin/hydroquinone/BHT, andarbutin/BHT/resveratrol. These antioxidant mixtures, which include threeantioxidants, may also optionally include a fourth antioxidant (which isdifferent from the first three) at an effective amount to further reducethe measured BROM value of the mixture relative to the sum of the BROMvalues of the individual antioxidants in the mixture at the sameconcentration, wherein the fourth antioxidant is chosen fromatorvastatin, BHT, hydroquinone, resveratrol, arbutin, uric acid andcombinations thereof.

Each of these two and three component mixtures of antioxidantsidentified by the Applicant may also yield a measured BROM value of theskin type product is greater than or equal to 0.2 sec/μg, or 0.25sec/μg, or 0.30 sec/μg, or 0.35 sec/μg, or 0.40 sec/μg, or 0.45 sec/μg,or 0.50 sec/μg, or 0.55 sec/μg, or 0.60 sec/μg, or 0.65 sec/μg, or 0.70sec/μg, or 0.75 sec/μg, there is sufficient protection from the harmfuleffects of UV radiation on the outer layers of the skin. Each of thesetwo and three component mixtures of antioxidants identified by theApplicant may also yield a phototoxic potential as measured by the 3T3Neutral Red Phototoxicity Test that is at least 5% lower, or 10% lower,or 15% lower, or 20% lower, or 25% lower, or 30% lower, or 35% lower, or40% lower, or 45% lower, or 50% lower relative to the control solution(non phototoxic and no antioxidants) at a solution concentration rangeof from 0.0001-0.002%. Alternatively, the concentration of the inventiveantioxidant mixtures in the finished skin type product may range from0.0002 to 0.0018%, or 0.0004 to 0.0016%, or 0.0006 to 0.0014%, or 0.0008to 0.0012%, or 0.0009 to 0.0011%.

The inventive antioxidants mixtures described above may be utilized in ahost of topically applied skin care products requiring UV protectivecapacity. Non-limiting exemplary skin care products include sunscreens,lip sticks, lip balms, skin whiteners, cosmetic products, pharmaceuticalproducts and dermatological products.

The following are examples of the present disclosure and are not to beconstrued as limiting.

EXAMPLES

BROM Measurements:

In Table 4 below, the BROM values of different combinations ofantioxidant compounds have been measured to determine antioxidantcapacity.

TABLE 4 BROM by Strenght Sample Re- Theoretical (R/TR − Size sultResult 1) *100 Ingredient Ratio [μg] Solvent [s/μg] [s/μg] [%]Hydroquinone 11  41-192 Water 0.55 0.46 18.65 Glucose 18 BHT 1 32-69 50%1.6 1.59 0.57 Hydroquinone 80 Ethanol Ascorbic 1 50% 1.96 2.8 −29.87Acid Ethanol Atorvastatin 1 100-140 Vit E 1 Acetate BHT 1 16-54 50% 2.21.96 12.33 Hydroquinone 40 Ethanol Atorvastatin 1 50% 2.76 4.12 −32.91Vit E 1  78-127 Ethanol Acetate Ascorbic 1 50-78 50% 3.31 4.24 −21.97Acid Ethanol Atorvastatin 1 BHT 1 40678 50% 8.04 7.36 9.19 Hydroquinone4 Ethanol Atorvastatin 1 31-55 50% 13 8.89 46.2 Uric Acid 2 EthanolAtorvastatin 2 29-40 50% 14.29 8.64 65.39 Uric Acid 1 Ethanol Arbutin 118598 Water 15.37 10.1 52.15 Hydroquinone 4 Ascorbic 1 35-55 50% 18.8618.1 4.4 Acid Ethanol Atorvastatin 1 BHT 2 Atorvastatin 1 24-37 50%19.19 8.79 118.41 Uric Acid 1 Ethanol BHT 1 40783 Water 19.61 23.05−14.92 Resveratrol 2 Arbutin 2 50% 22.58 18.26 23.64 BHT 1 16-38 EthanolHydroquinone 4 Atorvastatin 1 40-63 50% 26.56 20.21 31.38 BHT 1 EthanolAtorvastatin 1 40-67 50% 28.37 24.12 17.61 BHT 2 Ethanol Arbutin 2 50%32.77 32.84 −0.2 Atorvastatin 1 40771 Ethanol BHT 1 Arbutin 1 13-14 50%33.41 36.42 −8.28 BHT 2 Ethanol Arbutin 1 40648 Water 35 31.26 11.95Resveratrol 1 Arbutin 1 40680 50% 35.23 38.82 −9.27 BHT 1 EthanolArbutin 2 40716 50% 36.5 30.94 17.98 BHT 1 Ethanol Hydroquinone 1Arbutin 2 40711 50% 41.19 41.13 0.14 BHT 1 Ethanol Arbutin 2 40780 Water44.85 31.65 41.69 BHT 1 Resveratrol 2 Arbutin 4 40708 50% 45.4 42.9 5.82BHT 1 Ethanol

The results in Table 4 indicate that in some instances a synergisticincrease of antioxidant capacity occurs by combining two or moreantioxidants. This is indicated by an actual result over a theoreticalresult ((R/TR−1)*100) that is positive and greater than 1. The largestsynergistic increases are observed for different ratios of arbutin andhydroquinone, atorvastatin and uric acid, arbutin and BHT andatorvastatin with BHT.

In Table 5 below, the BROM values for different combinations of arbutinand hydroquinone were measured. The ratio of arbutin to hydroquinoneranged from 4:1 to 1:4. Throughout the range of ratios, the BROM valuesof the combinations of arbutin and hydroquinone yield a reducingcapacity that is significantly improved relative to the BROM values ofthe individual antioxidant ingredients. Hence, the combination ofarbutin and hydroquinone results in a synergistic improvement in BROMvalues over the range of ratios of arbutin to hydroquinone ranging from4:1 to 1:4. Arbutin and hydroquinone are substituted phenolics, whichare used as skin whitening agents. The combinations of substitutedphenolics with synergistic BROM values indicate their potentialeffectiveness as skin whitening combinations in cosmetic andpharmaceutical products.

TABLE 5 BROM Sample Re- Theoretical Size Sol- sult Result (R/TR −Ingredient Ratio [μg] vent [s/μg] [s/μg] 1) *100 [%] Arbutin 4 40766Water 44.72 36.76 21.64 Hydro- 1 quinone Arbutin 2 40828 Water 33.1330.73 7.79 Hydro- 1 quinone Arbutin 1 25-37 Water 25.28 15.89 59.08Hydro- 2 quinone Arbutin 1 18598 Water 15.37 10.1 52.15 Hydro- 4 quinone

Therefore any synergism present when using a combination of the two bestknown skin lightening agents i.e. arbutin and hydroquinone (HQ) mayallow one the ability to formulate a skin lightening product withmaximum efficacy and minimum toxicity. HQ reduces all three componentsof melanin production, that is, tyrosinase, melanin, and melanocytes.Hydroquinone is toxic to melanocytes. Arbutin inhibits tyrosinase;reduces melanin and is not toxic to melanocytes. Therefore, thesynergism between arbutin and hydroquinone will allow their use as moreeffective skin lightening agents with lower toxicity.

Another unexpected finding is the increased antioxidant capacity of ablend of two parts Arbutin by weight with one part of BHT and two partsof resveratrol. This blend shows an increase of approximately 40% morethan the contribution of the individual antioxidants. To test thehypothesis that Arbutin, BHT and resveratrol will be beneficial inprotecting the free radical damaging effects of UV radiation the threeantioxidants were prepared in a carrier of mixture of polysorbate 20,propylene glycol and ethanol. A control solution did not have anantioxidant (control). The free radical scavengers tested werearbutin/BHT (Sample 1 or S1), resveratrol (Sample 2 or S2), andresveratrol plus arbutin and BHT (Sample 3 or S3). The control and theS1, S2 and S3 solution were then exposed to the 3T3 Neutral RedPhototoxic Test described below and the BROM values were measured foreach. The BROM test results are included in Table 6 below. The resultsshow that each of S1, and S3 have high BROM values, while S2 only hasapproximately 1/10^(th) the BROM values of the other two. S1 and S3 withthe higher BROM value are effective in protecting murine fibroblastcells tested against the harmful effects of the high intensity simulatedsunlight source, while S2 is phototoxic to the cells.

Toxicological Testing:

In addition to BROM values, cytotoxicity and phototoxicity measurementswere made on the control, S1, S2 and S3 solutions. The 3T3 Neutral RedPhototoxicity Test (3T3 NRU PT) was used to assess toxicologicalperformance. It is a relatively new assay that was adopted by regulatoryagencies such as OECD and FDA as an accepted method for the assessmentof phototoxic potential of test substances. It is an in-vitrotoxicological assessment test used to determine cytotoxic andphoto(cyto)toxicity effect of a test article to murine fibroblasts inthe presence or absence of UVA light (99% UVA/1% UVB). The 3T3 NeutralRed Uptake Phototoxicity Assay (3T3 NRU PT) can be utilized to identifythe phototoxic effect of a test substance induced by the combination oftest substance and light and is based on the comparison of the cytotoxiceffect of a test substance when tested after the exposure and in theabsence of exposure to a non-cytotoxic dose of UVA/vis light.Cytotoxicity is expressed as a concentration dependent reduction of theuptake of the vital dye—Neutral Red. Substances that are phototoxicin-vivo after systemic application and distribution to the skin, as wellas compounds that could act as phototoxicants after topical applicationto the skin can be identified by the test. The reliability and relevanceof the 3T3 NRU PT have been evaluated and has been shown to bepredictive when compared with acute phototoxicity effects in vivo inanimals and humans.

To test the hypothesis that arbutin and BHT (both substituted phenolics)will either individually or in combinations be beneficial in protectingthe free radical damaging effects of UV radiation, different solutionswere prepared for testing as indicated in Table 8 below. The differentsolutions included a control (carrier with not antioxidants) as well astest solutions including the carrier with antioxidants for free radicalscavenging (S1:Control+arbutin/BHT, S2:Control+Resveratrol, andS3:Control+Resveratrol plus arbutin and BHT). The four solutions wereexposed to the BROM, and 3T3 Neutral Red Phototoxic Test and the resultsare indicated in Table 6 below with regard to BROM values and FIGS. 2-7with regard to cytotoxicity and photocytotoxicity results for SamplesS1, S2 and S3. The x-axis for each of the bar graphs are in units ofconcentration of the free radical scavenging antioxidants in thesolution. The results depicted FIGS. 2-7 indicate that certainantioxidant combinations in a carrier solution decrease the phototoxicpotential as measured by cytotoxicity and phototoxicity via the 3T3Neutral Red Phototoxicity Test by at least 5%, or 10%, or 15%, or 20%,or 25%, or 30%, or 35%, or 40%, or 45%, or 50% relative to the controlsolution (non phototoxic and no antioxidants) at a solutionconcentration range of from 0.0001-0.002%. The carrier solution for theantioxidant formulation may be a pharmaceutical, cosmetic ordermatological carrier for pharmaceutical, cosmetic or dermatologicalproducts.

TABLE 6 Sample Control S1 S2 S3 JR-42- JR-42- JR-42- JR-42- Designation24A 24C 26A 26C Arbutin 0.2 0.2 BHT 0.1 0.1 Resveratrol 0.2 0.2Polysorbate 0.5 0.5 0.5 0.5 20 Polypropylene 0.3 0.1 0.3 Glycol Ethanol99.2 70.1 99 99 Water 29 Total 100 100 100 100 BROM [s/ug] 0 0.166 0.0220.222 (of solution)

For both the range finding screen and the definitive test, Balb/c 3T3cells were seeded in the central 60 wells of duplicate 96-wellmicroplates per test article and maintained in culture for 24 hours. Thewell 96-well plates were then preincubated with eight differentconcentrations of each test article for one hour. After preincubation,one plate was irradiated with a dose of 5 J/cm**2. Solar Simulated Light(SSL, containing wavelengths in the UVA and visible regions with >99% ofUVB blocked out), while the duplicate plate was kept in the dark (noSSL). After UV irradiation, the treatment medium was replaced withculture medium and after 24 hours, cell viability was determined byneutral red uptake for 3 hours.

Test articles JR-42-24C sample 1, JR-42-26A sample 2, and JR-42-26C werephototested. Test articles JR-42-24C Sample 1, JR-42-26A Sample 2, andJR-42-26C Sample 3, provided by the Sponsor, were tested in a 3T3Neutral Red Uptake Phototoxicity Test using the Sponsor-provided vehicleJR-42-24A. Range finding screens were performed to determine theacceptable concentrations for the definitive test. Microsoft Excel® wasused to calculate the EC₅₀ values and Photo-irritant Factor (PIF) forthe test articles and the Chloropromazine (CPZ) positive control in boththe screen and the definitive test. Results of the definitive tests areshown in Table 7 below:

TABLE 7 Concentration EC₅₀ EC₅₀ + Test Article Range Tested No SSL SSLPIF JR-42-24C Sample 1 0.00032-0.004% >0.004%  >0.004% None JR-42-26ASample 2  0.0001-0.002% >0.002% >0.00071% >2.8 JR-42-26C Sample 3 NoSSL: 0.00046-0.005% + ND ND ND SSL: 0.00022-0.0032% CPZ Positive ControlNo SSL: 6.81-100 μg/ml + SSL: 0.22-3.16 μg/ml dose date: Mar. 10, 201119.592 μg/ml 0.394 μg/ml 49.67 dose date: Mar. 15, 2011 24.805 μg/ml0.725 μg/ml 34.19 dose date: Mar. 22, 2011 26.457 μg/ml 0.682 μg/ml38.80

From Table 7, it can be seen that the test article JR-42-24C Sample 1 isconsidered not to have phototoxic potential in the 3T3 Neutral RedUptake Phototoxicity Test. The range of antioxidants tested in sample 1would have been 300× more diluted than the concentration range tested(0.0003-0.004%). Test article JR-42-26A Sample 2 with 0.2% resveratrolis considered to have probable phototoxic potential in the concentrationrange tested (0.0001-0.002%) in the 3T3Neutral Red Uptake PhototoxicityTest. Test article sample 3, JR-42-26C has 0.2% resveratrol to which0.1% BHT and 0.2% arbutin were added. Test article JR-42-26C Sample 3was initially phototoxic in the 3T3Neutral Red Uptake PhototoxicityTest, but PIF values declined over time to non-phototoxic levels. Theresults indicated that although some phototoxicity is present theconcentration range tested, but increased 4 times more than the sampleswithout arbutin and BHT (sample 2). The conclusion obtained is theexpected effect of adding the arbutin and BHT to the somewhat phototoxicresveratrol. In addition, the phototoxic protection suggested by thecombination of arbutin and BHT indicates that even when exposed to theminimal erythemal dose (MED) of more than 250 times, the mili-molarpresence of the free radical scavengers is sufficient to protectfibroblast cells exposed 250 times the MED from phototoxic productsresulting from the degradation of chromophores as shown in the 3T3Neutral Red Phototoxic Test. As shown in Table 7, even though aphototoxic material is present, the photoxicity of the test sample isdecreased by a factor of 4.

Empirically it was determined that by the addition of 0.2% Arbutin and0.1% BHT to Sample 2, which then became Sample 3, the BROM value wasincreased from 0.02 sec/μg (Sample 2) to 0.2 sec/μg (Sample 3). This 10fold increase in BROM value proved to be effective in providingphototoxicity protection to the fibroblast cells using the 3T3 Test. Thenon-phototoxic results were also obtained for Sample 1 with a BROM valueof 0.166 sec/μg.

As an empirical guideline, in order to determine an effective protectionfactor for a finished product (FP), the Applicant has derived thefollowing relationship:BROM FP≧0.2 sec/μg % antioxidant needed for sunprotection=antioxidantcombination or individual BROM value÷100

The Applicant has discovered that when the BROM value of the finishedproduct is greater than or equal to 0.15 sec/μg, or 0.2 sec/μg, or 0.25sec/μg, or 0.30 sec/μg, or 0.35 sec/μg, or 0.40 sec/μg, or 0.45 sec/μg,or 0.50 sec/μg, or 0.55 sec/μg, or 0.60 sec/μg, or 0.65 sec/μg, or 0.70sec/μg, or 0.75 sec/μg, there is sufficient protection from the harmfuleffects of UV radiation on the outer layers of the skin.

For example, with an antioxidant combination of Arbutin at 0.2 wt. % andBHT at 0.1 wt. %, the Arbutin BROM value of 50 sec/μg×0.2% (multiply by0.002)=BROM of 0.1 and the .BHT BROM value of 30 sec/μg×0.1% (multiplyby 0.001)=BROM value 0.03. The antioxidant combination yields acalculated BROM value of approximately 0.13 (=0.1+0.03). Sample 1 has anactual BROM value of 0.166 sec/μg and therefore there is a slightsynergistic increase when Arbutin and BHT are combined. The aboverelationship is an empirical guideline and one should measure the actualBROM value for the finished product and also conduct photocytotoxicityand cytotoxicity testing as necessary.

In Table 8 below are BROM values for commercial products without theaddition of arbutin and BHT.

TABLE 8 Product BROM by Strength Sample Size Result [μg] Solvent [s/μg]Covergirl Nature Luxe 270 5700 Ethanol 0 Gloss Balm SPF15 CVS SportSunscreen SPF 71000 Ethanol 0 15 CVS Sport Sunscreen SPF 15000 50%Ethanol 0 30 CVS Sport Sunscreen SPF 64000 Ethanol 0 50 CVS Ultra DrySheer 25000 50% Ethanol 0 Lotion Sunscreen SPF 30 Neutrogena Age Shield18000 50% Ethanol 0 Face Sunblock SPF 55 Neutrogena Moisture Shine 18000Ethanol 0 Lip Soother Cooling Hydragel SPF20 Neutrogena Ultra Sheer24000 50% Ethanol 0 Dry-Touch Sunblock SPF30 Rimmel Moisture Renew 74016000 Ethanol 0 Auburn Breeze SPF18 Rimmel Moisture Renew 23000 Ethanol0 Cream Lipgloss SPF15 Australian Gold 19000-31000 50% Ethanol 0.003Clinique Dark Spot  430-1000 50% Ethanol 0.067 Corrector

The results in Table 8 indicate that the BROM values for commercialproducts are very low and therefore would be predicted to offer minimalprotection to the outer layers of the skin to the harmful effects of UVradiation.

In Table 9 below are BROM values for commercial products with theaddition of Arbutin and optionally BHT.

TABLE 9 BROM Alphabetical 1 of 1 Theoretical (BROM/ Antioxidant AddedSample Result BROM TBROM − 1) Name Amount [%] Size [μg] Solvent [s/μg][s/μg] *100 [%] Date Australian Gold Arbutin 0.5  700-2000 50% Ethanol0.390 0.311 25.4 Mar. 25, 2011 BHT 0.25 Clinique Dark Arbutin 1 150-1500 50% Ethanol 0.760 0.528 61.0 Dec. 2, 2010 Spot Corrector CVSSport Sun- Arbutin 0.5 1400-1900 50% Ethanol 0.33 0.308 8.4 May 2, 2011screen SPF 15 BHT 0.25 CVS Sport Sun- Arbutin 0.5 1700-2000 50% Ethanol0.29 0.308 −4.5 May 2, 2011 screen SPF 30 BHT 0.25 CVS Sport Sun-Arbutin 0.5 1600-2000 50% Ethanol 0.28 0.308 −8.9 May 4, 2011 screen SPF50 BHT 0.25

The results in Table 9 indicate that the Australian Gold, Clinique darkspot corrector and CVS sport sunscreen SPF 15 all showed an improvementin BROM values from 10 to 100 fold upon the addition of arbutin and BHT.

Lip Protection:

Full lips with an accentuated border have often been associated withbeauty and youth. It has been suggested that this is because the lipsoccupy both sides of the face and, with the smile, constitute a majorfocal point of overall facial beauty. Currently the only treatment forlip rejuvenation caused by UV radiation, smoking and other environmentalfactors is the use of sunscreens. The effect of UV radiation issignificantly more severe to the thin lip surface area, especially, ifthe lipstick, lip-gloss, lip or lip protectant product applied to thelip has chemical sunscreen includes. Sunscreen, especially on the lipsonly, protect three to five layers of kertinized cells that arecontinuously wiped and most likely swallowed when licking your lips. Thesunscreens on the lip are subject to the same photolytic decompositionas the skin. The phototoxic effect of DNA begins a process ofcarcinogenesis which may lead to cancerous tumor growths. To helpprevent the toxic cascade of events previously described for the skinthe same series of antioxidants should protect the lip area byscavenging the damaging free radicals present due to UV radiation.Attached are a series of formulas that include arbutin and BHT toincorporate generically in all lipsticks to help prevent the formationof cancerous cells. Below are 3 non-limiting exemplary formulas for lipproducts applying the inventive technology disclosed herein.

Example 1 Lip Treatment—Formula JR-39-90E

Carbowax 4000 (PEG-75) 25.00 Carbowax 8000 (PEG-180) 25.00 Carbowax 300(PEG-6) 29.70 Benzyl Alcohol 10.00 Z-Cote (Zinc Oxide) 10.00 Arbutin00.20 BHT 00.10 Total 100.00

Example 2 Lip Treatment—Formula JR-39-88B

Glycerin 81.20 Sodium Stearate 08.50 Benzyl Alcohol 10.00 Arbutin 00.20BHT 00.10 Total 100.00

Example 3 Lip Treatment—Formula JR-39-93B

Petrolatum 35.035 C13-15 Alkane 15.000 Dimethicone 02.000 Polyglyceryl-3behenate 03.000 BHT 00.100 Calcium Carbonate 10.000 Water 06.565Glycerin 22.000 Phenoxyethanol 01.000 Potassium Sorbate 00.100 Arbutin00.200 Total 100.000

Table 10 below gives the composition of two other lipstick formulas withantioxidants and with and without the addition of Arbutin and BHT.

TABLE 10 Lipstick Formula#1 with Anti-oxidants Active Ingredients:Octinoxate 7.50%   Octisalate 2.00%   Inactive Ingredients: Ricinus90.50%   Communis, 23.60%   Candelilla Cera, 4.50%   Octyldodecanol,20.75%   Ethylhexyl Methoxycinnamate 5.00%   Vp/Hexadecene Copolymer,2.50%   Myristyl Lactate, 2.00%   Caprylic/Capric Triglyceride, 20.00%  Lanolin, 2.00%   Ozokerite, 1.50%   Castor Oil/Ipdi Copolymer, 2.00%  Crambe Abyssinica, <1% Myristyl Myristate, <1% Ethylhexyl Salicylate,<1% Ethylhexyl Palmitate <1% Silica, Parfum, <1% Tocopherol, <1%Tribehenin, <1% Propylparaben, <1% Sorbitan Sesquioleate <1% Linalool,<1% Copernicia Cerifera, <1% Ascorbyl Palmitate, <1% PentaerythritylTetraisostearate, <1% Sorbitan Isostearate, <1% Hydroxycitronellal, <1%Hexyl Cinnamal, <1% Aqua, <1% Retinyl Palmitate, <1% Bht, <1% SilicaDimethyl Silylate, <1% Cinnamyl Alcohol, <1% Geraniol, <1% ButyleneGlycol, <1% Phenoxyethanol, <1% Palmitoyl Oligopeptide, <1% SodiumChondroitin Sulfate, <1% Caprylyl Glycol, <1% Atelocollagen, <1% Sodium<1% Hyaluronate, Methylparaben, <1% Hexylene Glycol, <1% Ethylparaben,<1% Butylparaben, <1% Isobutylparaben, <1% Synthetic Fluorphlogopite,<1% Calcium Aluminum Borosilicate <1% Tin Oxide <1% May Contain: MICA,CI 77891, CI 77491, CI 77492, CI 77499, CI 15850, CI 19140, CI 45410, CI15985, CI 17200, CI 42090, CI 75470.BROM

Time BROM Sample [mg] [s] [s/μg] 16 0 0Lipstick Formula #1 with Anti-Oxidants+1% Arbutin and 0.5% BHTBROM

Theo- Sam- retical (BROM/ ple Time BROM Arbutin BHT BROM TBROM − 1) [mg][s] [s/μg] [mg] [mg] [s/μg] *100 [%] 0.8339 375 0.45 0.00837 0.00390.607 −25.976 1.1221 497 0.443 0.01126 0.00525 0.607 −27.088 Aver- 0.450.61 −26.5 age:

For the above lipstick formulation with dozens of ingredients, acombination of 1% Arbutin to 0.5% BHT was added. The finished productBROM value of 0.45 secs/ug, which meets the criteria for finishedproducts to have BROM values above 0.2 secs/ug, which the Applicantestablished when testing for phototoxicity. Testing cosmetic andpharmaceutical products for safety and the use of the BROM antioxidantvalue for the measurement of free radical scavenging capacity in theupper layers of the skin is a novel application for this antioxidantcapacity assay.

All patents, test procedures, and other documents cited herein,including priority documents, are fully incorporated by reference to theextent such disclosure is not inconsistent with this invention and forall jurisdictions in which such incorporation is permitted.

When numerical lower limits and numerical upper limits are listedherein, ranges from any lower limit to any upper limit are contemplated.

Other Exemplary Embodiments:

In accordance with a first aspect of the disclosed embodiments, a methodof screening antioxidants for protecting the skin from UV radiation isprovided. The method includes applying the Briggs-Rauscher OxidantMethod (BROM) method to one or more antioxidants for predicting UVprotective capacity to the epidermal layer of the skin.

In accordance with a first aspect of the disclosed embodiments, themethod further includes optimizing the BROM method results to formulateantioxidant formulations for topically applied skin products requiringUV protection.

In accordance with a first sub-aspect of the disclosed embodiments,wherein the BROM value of the product is at least 0.2 sec/μg higher thanthe product without the optimized antioxidant formulation.

In accordance with a first aspect of the disclosed embodiments, whereinthe topically applied skin products are chosen from sunscreens, lipsticks, lip balms, skin whiteners, cosmetic products, pharmaceuticalproducts and dermatological products.

In accordance with a second aspect of the disclosed embodiments, anantioxidant formulation for use in sun screens, lip balms and othertopically applied skin care products for protecting the skin from UVradiation is provided. The antioxidant formulation including a mixtureof antioxidants in the skin care product including an effective amountof arbutin and BHT, wherein the ratio of arbutin to BHT in the skin careproduct ranges from 1:4 to 4:1, and wherein the measured BROM value ofthe antioxidant mixture is greater than the sum of the BROM values ofthe individual antioxidants in the mixture at the same concentration.

In accordance with a second aspect of the disclosed embodiments, theantioxidant formulation further including an effective amount ofhydroquinone, resveratrol, atorvastatin, uric acid and combinationsthereof.

In accordance with a second aspect of the disclosed embodiments, whereinthe measured BROM value of the product is greater than or equal to 0.2sec/μg.

In accordance with a second aspect of the disclosed embodiments, whereinthe phototoxic potential as measured by the 3T3 Neutral RedPhototoxicity Test is at least 5% lower than a control carrier solutionwithout the antioxidant formulation at a solution concentration range offrom 0.0001-0.002%.

In accordance with a third aspect of the disclosed embodiments, anantioxidant formulation for use in sun screens, lip balms and othertopically applied skin care products for protecting the skin from UVradiation is provided. The antioxidant formulation including a mixtureof antioxidants in the skin care product including an effective amountof atorvastatin and BHT, wherein the ratio of atorvastatin to BHT in theskin care product ranges from 1:2 to 1:1, and wherein the measured BROMvalue of the antioxidant mixture is greater than the sum of the BROMvalues of the individual antioxidants in the mixture at the sameconcentration.

In accordance with a third aspect of the disclosed embodiments, theantioxidant formulation further including an effective amount ofhydroquinone, resveratrol, arbutin, uric acid and combinations thereof.

In accordance with a third aspect of the disclosed embodiments, whereinthe measured BROM value of the product is greater than or equal to 0.2sec/μg.

In accordance with a first aspect of the disclosed embodiments, whereinthe phototoxic potential as measured by the 3T3 Neutral RedPhototoxicity Test is at least 5% lower than a control carrier solutionwithout the antioxidant formulation at a solution concentration range offrom 0.0001-0.002%.

In accordance with a fourth aspect of the disclosed embodiments, anantioxidant formulation for use in sun screens, lip balms and othertopically applied skin care products for protecting the skin from UVradiation is provided. The antioxidant formulation including a mixtureof antioxidants in the skin care product including an effective amountof arbutin and hydroquinone, wherein the ratio of arbutin tohydroquinone in the skin care product ranges from 1:4 to 4:1, andwherein the measured BROM value of the antioxidant mixture is greaterthan the sum of the BROM values of the individual antioxidants in themixture at the same concentration.

In accordance with a fourth aspect of the disclosed embodiments, theantioxidant formulation further including an effective amount of BHT,resveratrol, atorvastatin, uric acid and combinations thereof.

In accordance with a fourth aspect of the disclosed embodiments, whereinthe measured BROM value of the product is greater than or equal to 0.2sec/μg.

In accordance with a fourth aspect of the disclosed embodiments, whereinthe phototoxic potential as measured by the 3T3 Neutral RedPhototoxicity Test is at least 5% lower than a control carrier solutionwithout the antioxidant formulation at a solution concentration range offrom 0.0001-0.002%.

In accordance with a fifth aspect of the disclosed embodiments, anantioxidant formulation for use in sun screens, lip balms and othertopically applied skin care products for protecting the skin from UVradiation is provided. The antioxidant formulation including a mixtureof antioxidants in the skin care product including an effective amountof hydroquinone and BHT, wherein the ratio of hydroquinone to BHT in theskin care product ranges from 1:4 to 4:1, and wherein the measured BROMvalue of the antioxidant mixture is greater than the sum of the BROMvalues of the individual antioxidants in the mixture at the sameconcentration.

In accordance with a fifth aspect of the disclosed embodiments, theantioxidant formulation further including an effective amount ofarbutin, resveratrol, atorvastatin, uric acid, and combinations thereof.

In accordance with a fifth aspect of the disclosed embodiments, whereinthe measured BROM value of the product is greater than or equal to 0.2sec/μg.

In accordance with a fifth aspect of the disclosed embodiments, whereinthe phototoxic potential as measured by the 3T3 Neutral RedPhototoxicity Test is at least 5% lower than a control carrier solutionwithout the antioxidant formulation at a solution concentration range offrom 0.0001-0.002%.

In accordance with a sixth aspect of the disclosed embodiments, anantioxidant formulation for use in sun screens, lip balms and othertopically applied skin care products for protecting the skin from UVradiation is provided. The antioxidant formulation includes a mixture ofantioxidants in the skin care product including an effective amount ofatorvastatin and uric acid, wherein the ratio of atorvastatin to uricacid in the skin care product ranges from 0.5:1 to 2:1, and wherein themeasured BROM value of the antioxidant mixture is greater than the sumof the BROM values of the individual antioxidants in the mixture at thesame concentration.

In accordance with a sixth aspect of the disclosed embodiments, theantioxidant formulation further including an effective amount ofhydroquinone, resveratrol, arbutin, BHT, uric acid and combinationsthereof.

In accordance with a sixth aspect of the disclosed embodiments, whereinthe measured BROM value of the product is greater than or equal to 0.2sec/μg.

In accordance with a sixth aspect of the disclosed embodiments, whereinthe phototoxic potential as measured by the 3T3 Neutral RedPhototoxicity Test is at least 5% lower than a control carrier solutionwithout the antioxidant formulation at a solution concentration range offrom 0.0001-0.002%.

In accordance with a seventh aspect of the disclosed embodiments, anantioxidant formulation for use in sun screens, lip balms and othertopically applied skin care products for protecting the skin from UVradiation is provided. The antioxidant formulation includes a mixture ofantioxidants in the skin care product including an effective amount ofuric acid, atorvastatin and BHT, wherein the ratio of uric acid,atorvastatin and BHT in the skin care product ranges from 1:1:4 to4:1:1, and wherein the measured BROM value of the antioxidant mixture isgreater than the sum of the BROM values of the individual antioxidantsin the mixture at the same concentration.

In accordance with a seventh aspect of the disclosed embodiments, theantioxidant formulation further including an effective amount ofhydroquinone, resveratrol, arbutin, and combinations thereof.

In accordance with a seventh aspect of the disclosed embodiments,wherein the measured BROM value of the product is greater than or equalto 0.2 sec/μg.

In accordance with a seventh aspect of the disclosed embodiments,wherein the phototoxic potential as measured by the 3T3 Neutral RedPhototoxicity Test is at least 5% lower than a control carrier solutionwithout the antioxidant formulation at a solution concentration range offrom 0.0001-0.002%.

In accordance with an eighth aspect of the disclosed embodiments, anantioxidant formulation for use in sun screens, lip balms and othertopically applied skin care products for protecting the skin from UVradiation is provided. The antioxidant formulation includes a mixture ofantioxidants in the skin care product including an effective amount ofarbutin and resveratrol, wherein the ratio of arbutin to resveratrol inthe skin care product ranges from 1:4 to 4:1, and wherein the measuredBROM value of the antioxidant mixture is greater than the sum of theBROM values of the individual antioxidants in the mixture at the sameconcentration.

In accordance with an eighth aspect of the disclosed embodiments, theantioxidant formulation further including an effective amount ofhydroquinone, BHT, atorvastatin, uric acid and combinations thereof.

In accordance with an eighth aspect of the disclosed embodiments,wherein the measured BROM value of the product is greater than or equalto 0.2 sec/μg.

In accordance with an eighth aspect of the disclosed embodiments,wherein the phototoxic potential as measured by the 3T3 Neutral RedPhototoxicity Test is at least 5% lower than a control carrier solutionwithout the antioxidant formulation at a solution concentration range offrom 0.0001-0.002%.

In accordance with an ninth aspect of the disclosed embodiments, anantioxidant formulation for use in sun screens, lip balms and othertopically applied skin care products for protecting the skin from UVradiation is provided. The antioxidant formulation includes a mixture ofantioxidants in the skin care product including an effective amount ofBHT and resveratrol, wherein the ratio of BHT to resveratrol in the skincare product ranges from 1:4 to 4:1, and wherein the measured BROM valueof the antioxidant mixture is greater than the sum of the BROM values ofthe individual antioxidants in the mixture at the same concentration.

In accordance with an ninth aspect of the disclosed embodiments, theantioxidant formulation further including an effective amount ofhydroquinone, arbutin, atorvastatin, uric acid and combinations thereof.

In accordance with an ninth aspect of the disclosed embodiments, whereinthe measured BROM value of the product is greater than or equal to 0.2sec/μg.

In accordance with an ninth aspect of the disclosed embodiments, whereinthe phototoxic potential as measured by the 3T3 Neutral RedPhototoxicity Test is at least 5% lower than a control carrier solutionwithout the antioxidant formulation at a solution concentration range offrom 0.0001-0.002%.

In accordance with a tenth aspect of the disclosed embodiments, anantioxidant formulation for use in sun screens, lip balms and othertopically applied skin care products for protecting the skin from UVradiation is provided. The antioxidant formulation includes a mixture ofantioxidants in the skin care product including an effective amount ofatorvastatin and hydroquinone, wherein the ratio of atorvastatin tohydroquinone in the skin care product ranges from 1:4 to 4:1, andwherein the measured BROM value of the antioxidant mixture is greaterthan the sum of the BROM values of the individual antioxidants in themixture at the same concentration.

In accordance with a tenth aspect of the disclosed embodiments, theantioxidant formulation further including an effective amount of BHT,resveratrol, arbutin, uric acid and combinations thereof.

In accordance with a tenth aspect of the disclosed embodiments, whereinthe measured BROM value of the product is greater than or equal to 0.2sec/μg.

In accordance with a tenth aspect of the disclosed embodiments, whereinthe phototoxic potential as measured by the 3T3 Neutral RedPhototoxicity Test is at least 5% lower than a control carrier solutionwithout the antioxidant formulation at a solution concentration range offrom 0.0001-0.002%.

In accordance with an eleventh aspect of the disclosed embodiments, anantioxidant formulation for use in sun screens, lip balms and othertopically applied skin care products for protecting the skin from UVradiation is provided. The antioxidant formulation includes a mixture ofantioxidants in the skin care product including an effective amount ofarbutin, hydroquinone and BHT, wherein the ratio of arbutin,hydroquinone and BHT in the skin care product ranges from 2:1:1 to2:1:4, and wherein the measured BROM value of the antioxidant mixture isgreater than the sum of the BROM values of the individual antioxidantsin the mixture at the same concentration.

In accordance with an eleventh aspect of the disclosed embodiments, theantioxidant formulation further including an effective amount ofatorvastatin, resveratrol, uric acid and combinations thereof.

In accordance with an eleventh aspect of the disclosed embodiments,wherein the measured BROM value of the product is greater than or equalto 0.2 sec/μg.

In accordance with an eleventh aspect of the disclosed embodiments,wherein the phototoxic potential as measured by the 3T3 Neutral RedPhototoxicity Test is at least 5% lower than a control carrier solutionwithout the antioxidant formulation at a solution concentration range offrom 0.0001-0.002%.

In accordance with a twelfth aspect of the disclosed embodiments, anantioxidant formulation for use in sun screens, lip balms and othertopically applied skin care products for protecting the skin from UVradiation is provided. The antioxidant formulation includes a mixture ofantioxidants in the skin care product including an effective amount ofarbutin, BHT and resveratrol, wherein the ratio of arbutin, BHT andresveratrol in the skin care product ranges from 1:1:1 to 2:1:2, andwherein the measured BROM value of the antioxidant mixture is greaterthan the sum of the BROM values of the individual antioxidants in themixture at the same concentration.

In accordance with a twelfth aspect of the disclosed embodiments,further including an effective amount of atorvastatin, hydroquinone,uric acid and combinations thereof.

In accordance with a twelfth aspect of the disclosed embodiments,wherein the measured BROM value of the product is greater than or equalto 0.2 sec/μg.

In accordance with a twelfth aspect of the disclosed embodiments,wherein the phototoxic potential as measured by the 3T3 Neutral RedPhototoxicity Test is at least 5% lower than a control carrier solutionwithout the antioxidant formulation at a solution concentration range offrom 0.0001-0.002%.

What is claimed is:
 1. An antioxidant formulation for use in sunscreens, lip balms and other topically applied skin care products forprotecting the skin from UV radiation comprising: a mixture ofantioxidants in the skin care product including an effective amount ofatorvastatin and uric acid, wherein the ratio of atorvastatin to uricacid in the skin care product ranges from 1:2 to 2:1, and wherein themeasured BROM value of the antioxidant mixture is greater than the sumof the BROM values of the individual antioxidants in the mixture at thesame concentration.
 2. The antioxidant formulation of claim 1, furtherincluding an effective amount of hydroquinone, resveratrol, arbutin,butylated hydroxytoluene (BHT), and combinations thereof.
 3. Theantioxidant formulation of claim 1, wherein the measured BROM value ofthe product is greater than or equal to 0.2 sec/μg.
 4. The antioxidantformulation of claim 1, wherein the phototoxic potential as measured bythe 3T3 Neutral Red Phototoxicity Test is at least 5% lower than acontrol carrier solution without the antioxidant formulation at asolution concentration range of from 0.0001-0.002%.